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R E S E A R C H A R T I C L E

Open Access

Chromosomal imbalances are uncommon in

chagasic megaesophagus

Marilanda F Bellini

1,2

, Antonio J Manzato

3

, Ana E Silva

1*

, Marileila Varella-Garcia

2

Abstract

Background:Chagas’disease is a human tropical parasitic illness and a subset of the chronic patients develop megaesophagus or megacolon. The esophagus dilation is known as chagasic megaesophagus (CM) and one of the severe late consequences of CM is the increased risk for esophageal carcinoma (ESCC). Based on the association between CM and ESCC, we investigated whether genes frequently showing unbalanced copy numbers in ESCC were altered in CM by fluorescence in situ (FISH) technology.

Methods:A total of 50 formalin-fixed, paraffin-embedded esophageal mucosa specimens (40 from Chagas megaesophagus-CM, and 10 normal esophageal mucosa-NM) were analyzed. DNA FISH probes were tested for

FHIT,TP63, PIK3CA,EGFR, FGFR1, MYC,CDKN2A, YES1andNCOA3genes, and centromeric sequences from

chromosomes 3, 7 and 9.

Results:No differences between superficial and basal layers of the epithelial mucosa were found, except for loss of copy number ofEGFRin the esophageal basal layer of CM group. Mean copy number ofCDKN2A andCEP9 and frequency of nuclei with loss ofPIK3CAwere significantly different in the CM group compared with normal mucosa and marginal levels of deletions inTP63, FHIT, PIK3CA, EGFR, CDKN2A, YESand gains atPIK3CA, TP63, FGFR1, MYC,

CDNK2AandNCOA3were detected in few CM cases, mainly with dilation grades III and IV. All changes occurred at

very low levels.

Conclusions:Genomic imbalances common in esophageal carcinomas are not present in chagasic

megaesophagus suggesting that these features will not be effective markers for risk assessment of ESCC in patients with chagasic megaesophagus.

Background

Chagas’ disease is a human tropical parasitic disease which occurs in the Americas, particularly in South America. It affects 16 to 18 million people in tropical and subtropical countries of Latin America [1]; in Brazil, the number of cases has reached 6 million [2]. During the chronic phase, 6 to 7% of chagasic patients develop mega syndromes represented by muscular hypertrophy and dilation of the esophagus or colon, in consequence of destruction of the myoenteric and submucous plexus by the protozoan Trypanosoma cruzi[3]. The digestive forms, megaesophagus or megacolon may be observed in advanced stages of the disease [3].

Megaesophagus is consequence of achalasia character-ized by the destruction or lack of intramural nerve

plexus, which determines the absence of peristalsis and lack of openness of the lower esophageal sphincter in response to swallowing. In consequence, food retention or esophageal stasis occurs, leading to the appearance of chronic esophagitis, acanthosis, paraceratose and leuko-plakia, possibly pre-cancerous lesions [3]. One of the severe late consequences of chagasic megaesophagus is the increased risk (3% to 8%) of developing esophageal squamous cell carcinoma (ESCC) compared to when megaesophagus is not present [4-6]. Also, ESCC devel-ops in chagasic megaesophagus patients at a younger age than in those without this disease [5]. The detection of cancer in these patients is difficult because the symp-toms are hidden by the severe dysphagia caused by megaesophagus [7]. The diagnosis is frequently late, when the patient is in advanced stage, resulting in a poor prognosis [5].

* Correspondence: anabete@ibilce.unesp.br

1UNESP, São Paulo State University, Department of Biology, Campus São José do Rio Preto, SP, Brazil

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ESCC has been reported as the ninth most common malignancy and ranks the sixth most frequent cause of death worldwide, but its incidence varies largely among regions [8]. Approximately 16,470 new cases of esopha-geal carcinoma were expected in the US population in 2009 [9]. The Brazilian National Institute of Research in Cancer (INCA) reported that esophageal cancer was the sixth in the cancer rank mortality in 2000 with 5,307 deaths. The estimated incidence in 2008 was 10550 new cases with an incidence per 100 thousand individuals varying among geographical areas from 1.04 to 19.07 in males and 0.39 to 7.58 in females [8].

In ESCC, molecular cytogenetic techniques have shown common occurrence of unbalanced genomic regions involved in amplification of oncogenes and dele-tion of tumor suppressor genes [10,11]. However, stu-dies in benign esophageal lesions with precancerous potential as megaesophagus are scarce. This study used FISH technique to investigate chagasic megaesophagus for genomic status of genes frequently unbalanced in ESCC with the goal of identifying potential markers of risk to cancer. The selected targets included FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1 and NCOA3 genes, all of which have been reported as occurring in significantly abnormal numbers in ESCC [12-19].

Methods

Subjects

Formalin-fixed, paraffin-embedded (FFPE) esophageal mucosa was obtained from 40 patients with diagnosis of chagasic megaesophagus (CM) who underwent middle and distal esophageal biopsies from 2000 to 2007 at the Hospital de Base (São José do Rio Preto, SP, Brazil). The study was approved by the Institution Research Ethical Committee (CEP) and by the Brazilian National Research Ethics Committee (CONEP), and written informed consent was obtained from all patients. Among the 40 CM patients, the mean age was 62.5 years (range: 40 - 83 years) and 20 were male; 29 were never-alcoholics, 3 former-alcoholics (≥5 years of absti-nence) and 8 current alcoholics; 18 were never-smokers, 6 former-smokers (≥5 years of quitting) and 16 were current smokers. The megaesophagus diagnosis was made by physical examination, and radiologic and endo-scopic study of esophageal motility by manometry [3]. The dilation grade of megaesophagus was classified as I to IV, based on the retention of contrast, diameter, toni-city of the lower sphincter and the length of the esopha-gus body [20]. This study cohort included 5 patients with megaesophagus grade I, 7 with grade II, 17 with grade III and 11 with grade IV. The CM group was fol-lowed for 2 to 4 years (median follow up = 2,8 years)

and no one developed ESCC, probably due to the short follow up of patients.

The control group (NM) was composed by 10 health subjects who were submitted to endoscopy under suspi-cion of dyspepsia, but the histopathological analysis has shown normal esophageal mucosa. In this group, the mean age was 42.4 (range: 26 - 67 years); 3 individuals were male and 7 female; 6 were never-alcoholics and four current alcoholics; 7 were never smokers and 3 current smokers.

Fluorescence In Situ Hybridization (FISH)

Serial 4 μm-thick sections were cut from paraffin-embedded blocks and mounted in glass slides pre-trea-ted with 3-aminopropyl-triethoxysilane/acetone solution. Specimens were incubated at 56°C overnight, deparaffi-nized in Citrisolv washes (Fisher Brand, Cat #22-143975) (three times for 10 min each), and dehydrated in 100% ethanol. After incubation in 2 × SSC (Sodium chloride, sodium citrate solution, pH 7.0) at 75°C, sec-tions were digested with proteinase K (0.25 mg/ml in 2 × SSC, pH 7.0) at 45°C, rinsed in 2 × SSC (pH 7.0) at room temperature for 5 min and dehydrated in an etha-nol series.

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Fluorescence signals were scored in epifluorescence microscope using single band filters for DAPI, FITC (fluorescein), and Texas red, double-band pass filter (FITC and Texas red) and triple-band pass filter (DAPI, FITC, and Texas Red). Histological areas previously selected in the HE-stained (hematoxylin and eosin) sec-tions were identified in the hybridized slides. Signals were scored in 100 epithelial nuclei per specimen, in at least four distinct areas: 50 nuclei in the superficial layer (larger cells, closer to the lumen) and 50 nuclei in the basal layer (darker HE-stained nuclei, smaller cells, clo-ser to the muscular layer). The scoring was performed in both layers to check for differences between prolifer-ate activity, since basal layer cells are in high prolifera-tive activity, whereas the superficial layer cells are more mature. For 3 genes, FHIT, EGFR andCDKN2A, the experiments were performed including centromeric sequences of carrier chromosomes as an internal con-trol, respectively chromosomes 3, 7 and 9. This design was necessary forFHITandCDKN2Asince these genes are known for their loss in cancer specimens [21,22]. For each FISH probe set, tissue sections from two nor-mal subjects were used as control.

Statistical Analysis

Descriptive statistics were calculated using an Microsoft Excel macro template previously validated including mean copy number per cell, standard deviation and fre-quency of cells with 1, 2 and ≥3 copies of each DNA target tested, as well as the ratio for gene/control probe when applicable. The superficial and basal layers were compared by t-student paired test, and between the experimental groups were compared by t-student unpaired test; in both cases confidence level was estab-lished as 0.05 [23]. Association between gene status with age, gender, life style factors (smoking and alcoholism), and megaesophagus grade in the CM group was com-pared by ANOVA and the comparisons for losses and gains, and aneusomies, between the groups, were done by c2test. One exploratory analysis using three-dimen-sional plots was performed for the percentages of genes status classes.

Results

The descriptive indexes (mean and standard deviation) for the tested genes (FHIT, PIK3CA, TP63, EGFR, FGFR1, MYC, CDKN2A, YES1, NCOA3) and centro-meric sequences (chromosomes 3, 7 and 9 evaluated respectively with the genesFHIT, EGFRandCDKN2A), and the results of the statistical analyses for comparison of mean copy numbers between the superficial and basal layers in each group are presented in Table 1. The mean values in the CM group ranged from 1.49 to 1.78 in the superficial layer and from 1.45 to 1.79 in the

basal layer. In the NM group, the mean frequencies ran-ged from 1.35 to 1.77 in the superficial layer and from 1.61 to 1.79 in the basal layer. Superficial and basal layers of the esophageal mucosa did not show significant differences, excepted for EGFR in the CM group (p = 0.008) that showed lower copy number in the basal layer. Results from both layers were combined for com-parison of mean copy numbers per cell of the each molecular target and no difference was detected between CM and NM. However, the copy number of CDKN2Awas significantly lower (p < 0.0001) in the CM group compared to NM, and similar results were observed for the corresponding centromere 9 (CEP9) (p = 0.0002).

In another investigative approach, frequencies of nuclei with normal (2 copies) or loss (<2 copies) and gain (>2 copies) for each gene were compared (Table 2), and only for PIK3CAthere were higher frequencies of cells with copy number loss in the CM group compared to NM (36.4% vs. 28.5%). Statistical analysis was not performed for gain due to the scarcity of cells with this pattern. Aneusomies involving centromeres of chromo-somes 3, 7, and 9 were also investigated (Table 2). The CM group showed significantly increased frequencies of cells with copy number loss of centromeres of chromo-somes 7 and 9 (51% and 44%, respectively) in compari-son to the NM group (28% and 23%, respectively). The CM group also showed numerically higher frequencies of cells with copy number gain of centromeres of chro-mosomes 7 and 9 (1.68% and 10.38%, respectively) than the NM group (0.50% and none, respectively), although the numbers were too small for statistical analyses.

The gene/centromere ratios for FHIT/CEN3, EGFR/ CEP7 and CDKN2A/CEP9, the probe sets including matched targets in the study, were all balanced (close to 1) and differences were not detected between the mean copy number of each target within each group (Table 3). These results suggest that loss of FHIT, EGFR and CDKN2A was accompanied by loss of their respec-tive chromosomes.

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No significant association was detected in CM group between mean copy number per cell of each DNA target tested and parameters such as age, gender and the life style factors tobacco smoking and alcoholism although the small size of each subset impaired robust conclu-sion. Similarly, no significant differences were observed on mean copy number per cell of each genomic target according the dilation grades of megaesophagus (data not shown). However, the CM cases identified with genomics deletions and gains in the three-dimensional plots were mostly classified as grades III and IV, sug-gesting that chromosomal imbalances are more likely to occur in more advanced grades.

Discussion

Esophageal squamous cell carcinoma is one of the most prevalent cancers worldwide and has multifactorial ori-gin, in which environment factors, especially alcohol ingestion and smoking, play significant role [10,24]. Sev-eral genetic alterations are also associated with ESCC,

such as chromosomal changes, allelic deletions, activa-tion of oncogenes and inactivaactiva-tion of tumor suppressor genes [10,11]. In tropical and subtropical countries of Latin America such as Brazil, the risk of ESCC may also be related to megaesophagus due to Chagas’disease. The period between onset of symptoms related to acha-lasia and detection of cancer ranges from 17 to 28 years [4]. In the current study no patient developed esopha-geal carcinoma, which can also be due to insufficient follow up.

This study showed that genomic copy number changes were not common events in megaesophagus. Among the tested 12 DNA targets mapped to 6 distinct chromosomes, there was only marginal differences between specimens from chagasic megaesophagus and normal esophageal mucosa. The only statistically signifi-cant difference in copy number between groups involved CDKN2A. Loss of function of CDKN2A (also called MTS-1 or P16) prevents the blocking of G1 phase and appears to be a necessary event for the progression of Table 1 Mean copy number per cell for the 12 DNA targets tested in esophageal mucosa of chagasic megaesophagus and normal mucosa groups.

CM NM

Targets Mucosa Layers Mucosa Layers t-Student

CM × NM

Superficial Basal Total Superficial Basal Total

Mean ± SD Mean ± SD

FHIT 1.77 ± 0.08 1.78 ± 0.08 1.76 ± 0.08 1.35 ± 0.49 1.75 ± 0.04 1.74 ± 0.04 nsP = 0.7803

Range: 1.56-1.90 Range: 1.71-1.77

PIK3CA 1.76 ± 0.09 1.78 ± 0.08 1.77 ± 0.07 1.70 ± 0.09 1.78 ± 0.11 1.74 ± 0.01 nsP = 0.6982

Range: 1.56-1.96 Range: 1.64-1.86

TP63 1.77 ± 0.11 1.78 ± 0.09 1.77 ± 0.09 1.70 ± 0.09 1.78 ± 0.11 1.74 ± 0.01 nsP = 0.5304

Range: 1.56-2.06 Range: 1.64-1.86

EGFR 1.52 ± 0.12a 1.45 ± 0.15b 1.49 ± 0.13a, b 1.60 ± 0.00 1.74 ± 0.01 1.67 ± 0.01 nsP = 0.3700

Range: 1.25-1.80 Range: 1.60-1.75

FGFR1 1.78 ± 0.01 1.76 ± 0.10 1.77 ± 0.08 1.63 ± 0.01 1.70 ± 0.03 1.67 ± 0.03 nsP = 0.2200

Range: 1.56-2.22 Range: 1.62-1.72

MYC 1.76 ± 0.09 1.75 ± 0.09 1.76 ± 0.08 1.66 ± 0.01 1.74 ± 0.02 1.70 ± 0.01 nsP = 0.5396

Range: 1.58-1.77 Range: 1.69-1.70

CDKN2A 1.49 ± 0.08 1.50 ± 0.07 1.50 ± 0.06 1.76 ± 0.14 1.72 ± 0.01 1.74 ± 0.07 * P < 0.0001

Range: 1.34-1.59 Range: 1.69-1.79

YES1 1.67 ± 0.13 1.67 ± 0.12 1.67 ± 0.11 1.70 ± 0.08 1.68 ± 0.13 1.69 ± 0.07 nsP = 0.8099

Range: 1.37-1.99 Range: 1.58-1.75

NCOA3 1.65 ± 0.11 1.64 ± 0.11 1.64 ± 0.10 1.66 ± 0.11 1.61 ± 0.01 1.64 ± 0.06 nsP = 0.7656

Range: 1.37-1.80 Range: 1.59-1.68

Centromere 3 1.77 ± 0.08 1.79 ± 0.07 1.77 ± 0.07 1.71 ± 0.07 1.76 ± 0.08 1.74 ± 0.08 nsP = 0.6638

Range: 1.56-1.90 Range: 1.68-1.79

Centromere 7 1.53 ± 0.12 1.50 ± 0.16 1.52 ± 0.13 1.67 ± 0.01 1.79 ± 0.04 1.73 ± 0.21 nsP = 0.1235

Range: 1.26-1.53 Range: 1.76-1.82

Centromere 9 1.53 ± 0.09 1.53 ± 0.09 1.53 ± 0.07 1.77 ± 0.15 1.77 ± 0.04 1.77 ± 0.10 * P = 0.0002

Range: 1.33-1.65 Range: 1.70-1.84

CM, chagasic megaesophagus; NM, normal mucosa; SD, standard-deviation; Statistical Analysis:t-Student; *, P < 0.05;ns

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pre-cancerous cells to malignancy [25,26]. Genetic and epigenetic alterations inCDKN2Ahave been reported in early stages of esophageal carcinogenesis [27-30]. In pri-mary esophageal carcinomas and cell lines, deletions and point mutations in CDKN2A gene have been reported in 16% to 82% of cases [22,31], and hyper-methylation of promoter region in 20% to 88% of ESCC and precancerous lesions [30,32]. The current study detected deletion ofCDKN2Aand CEP 9 sequences in

CM compared with NM group. In agreement with these findings, patients with idiopathic achalasia and chagasic megaesophagus, with or without esophageal carcinoma, showed reductions in expression of p16 protein [7] and Belliniet al. [33] observed a marginal decrease in p16 protein expression in chagasic megaesophagus. Addi-tionally, recent subset analyses by our group did not find mutations in CDKN2A(exons 1 and 2) and FHIT (exons 5 and 7) genes, suggesting these events are uncommon in CM [34], and have detected copy number changes of chromosomes 7, 11 and 17 and TP53 dele-tion by FISH [35].

The tumor suppressor geneFHIT (3p14.2) is deregu-lated during the development of ESCC. Deletion in both FHIT alleles results in failure in the transcript [36] and consequent absence or reduction of Fhit protein, which act in the cell cycle regulation and apoptosis. Genetic and epigenetic alterations of FHITare associated with development of several cancer types [37,38]. Loss of het-erozygosity (LOH) in areas of the 3p14-p21 bands has also been reported in low-grade dysplasia and postulated as early events in esophageal carcinogenesis [39]. While hypermethylation of FHITgene has been described in 33% to 69.4% of ESCC cases, it is also known that dele-tion and loss of protein expression are frequent in eso-phageal carcinoma [32,36]. Deletion of FHITobserved in a single megaesophagus specimen in the present study may associate with early onset of genetic chances in these lesions, but it is worth to note that no signifi-cant decrease in the level of Fhit protein expression have been detected in chagasic megaesophagus [33].

All other tested genes are categorized as oncogenes and have been previously shown to associate with ESCC, in which they are commonly amplified [12-19]. However, abnormal copy number patterns were only seen in few individuals in the Chagasic megasophagus Table 2 Frequencies of cells with loss, normal status and

gain in copy numbers of the target in chagasic megaesophagus and normal mucosa groups.

<2 copies per cell 2 copies per cell

>2 copies per cell

Target CM NM c2test CM NM CM NM

FHIT

N 20 2 P = 10.672ns 2478 144 6 3 % 0.79 1.34 98.96 96.64 0.24 2.04

PIK3CA

N 898 57 P = 0.000* 1530 139 35 4 % 36.39 28.50 61.99 69.50 1.42 2.00

TP63

N 885 58 P = 2.579ns 2878 136 37 6 % 23.29 29.00 75.74 68.00 0.97 3.00

EGFR

N 298 12 P = 1.067ns 3433 187 171 1 % 7.59 6.00 88.03 93.50 4.38 0.50

FGFR1

N 928 68 P = 4.172ns 2833 121 46 1 % 24.38 34.00 74.41 60.50 1.21 0.50

MYC

N 918 62 P = 4.812ns 2849 135 33 2 % 24.00 31 75.00 67.50 0.01 1.00

CDKN2A

N 183 6 P = 17.702ns 3649 194 68 0 % 4.69 3.00 93.56 97.00 1.74 0.00

YES1

N 1381 62 P = 2.171ns 2407 136 12 2 % 36.34 31.00 63.34 68.00 0.32 1.00

NCOA3

N 45 8 P = 3.627ns 2366 130 8 0 % 1.86 5.80 97.81 94.20 0.33 0.00 CEN3

N 683 54 P = 0.470ns 2510 145 7 1 % 21.0 27.0 78.44 72.5 0.56 0.50 CEP7

N 1637 56 P = 0.00+* 1509 1143 54 1 % 51.16 28.0 47.16 71.50 1.68 0.50 CEP9

N 1400 46 P = 0.00+* 1468 154 332 0 % 43.75 23.0 45.87 77.0 10.38 0.00

CM, chagasic megaesophagus; NM, normal mucosa; SD, standard-deviation; Statistical Analysis:t-Student; *, P < 0.05;ns

, P > 0.05;a, b , P = 0.008.

Table 3 Comparison between matched gene and centromere targets, in chagasic megaesophagus and normal mucosa groups.

Gene-Centromere CM NM

Mean ± SD Ratio Mean ± SD Ratio

FHIT 1.77 ± 0.08 1.00 1.74 ± 0.04 1.00 CEN3 1.77 ± 0.08 1.74 ± 0.08

t-Student ns ns

EGFR 1.49 ± 0.12 0.98 1.67 ± 0.01 0.96 CEP7 1.51 ± 0.13 1.73 ± 0.02

t-Student ns ns

CDKN2A 1.51 ± 0.09 1.00 1.74 ± 0.07 0.98

CEP9 1.54 ± 0.10 1.77 ± 0.10

t-Student ns ns

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cohort and involved both gains and losses. One example is PIK3CA, which encodes the catalytic subunit that uses ATP to phosphorylate phosphatidylinositol, a gene that is frequently amplified in ESCC [40-42]. Yen et al. [40] determined in FISH assays thatPIK3CAwas ampli-fied in cell lines and primary tumors and detected a positive correlation between amplification and tumor size, lymph node metastasis and clinical stage. Yang et al. [41] showed that PIK3CA gene amplification was

highly correlated with protein overexpression, support-ing amplification as a major mechanism for overexpres-sion. Nevertheless, in this study PIK3CAwas lost rather than amplified in the CM specimens as a group while involved in marginal level of loss two specimens and gained in two other specimens.

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plays in the development and maintenance of stratified epithelial tissues has been described in early stage of ESCC carcinogenesis but down-regulated in advanced [18,43]. Amplification of the membrane receptor genes EGFRandFGFR1has been reported in various cancers including ESCC [45,46], and this phenomenon has been suggested to be a poor prognostic factor in solid tumors [47]. In ESCC, co-expression of bothaFGF andFGFR1 was associated with larger tumor area and worse prog-nosis which suggests that the membrane receptor may promote proliferation of esophageal cancer cells in an angiogenesis-independent and autocrine manner and may contribute to rapid recurrence after esophageal resection [47].

MYC is a transcription factor that binds E-boxes as a heterodimer with Max in about 15% of all genes, and recruits co-activators to regulate gene transcription. MYCis regulated in part through mitogenic stimuli and is activated constitutively in cancer cells through gene amplification, chromosomal translocation, point muta-tion and mitogenic stimulamuta-tion [48].MYCwas found to be amplified in ESCC cell lines and in primary tumors [14].YES1 codes a protein with tyrosine kinase activity

that has been previously associated with esophageal car-cinogenesis [15,42]. Similarly, the NCOA3gene encod-ing a nuclear receptor coactivator that interacts with nuclear hormone receptors to enhance their transcrip-tional activator function was found frequently overex-pressed and also amplified in 5 to 15% of ESCCs [49-51].

Although is well established that megaesophagus pre-ferentially affects males between the second and fourth decades of life [3], this study has not detected relation-ship between copy numbers of each tested gene and parameters such as age, gender, life style factors (tobacco smoking and alcoholism) and megaesophagus grade in the CM group. Nevertheless these findings may have been impacted by the limited number of samples in each category.

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with the production of N-nitrous compounds, which has potent action in esophageal carcinogenesis [52]. High grade megaesophagus present higher dilation and eso-phageal stasis associated with bacterial proliferation, thus the data suggest that the level of dilation and inflammation of megaesophagus could promote genetic damages and could be related to increased risk of tumor development [4,7,53].

Conclusion

In conclusion, among 40 chagasic megaesophagus com-prehensively investigated with a panel of FISH probes, copy number gain was only displayed by three speci-mens for MYC, by one specimen for FGFR1 and one specimen forNCOA3. Loss ofFHITandYES1was seen in one specimen and of EGFR in another. For the remainder genesCDKN2A, PIK3CAand TP63, from one to four specimens each showed copy number gain or loss. In every case the difference was only marginal therefore not providing strong support to a biological impact. These findings show that imbalances involving the genomic regions encompassing gene sequences relevant in esophageal carcinogenesis were not found as significant events in chagasic megaesophagus. Conse-quently, genomic imbalances are not promising markers for assessment of ESCC risk in chagasic megaesophagus.

List of abbreviations

aFGF: endothelial cell growth factor, alpha; ANOVA: Analysis of Variance; BAC: Bacterial Artificial Chromo-somes; Cat. #: Catalog number;CDKN2A: cyclin-depen-dent kinase inhibitor 2A (melanoma, p16: inhibits CDK4); CEN3: Centromere 3; CEP: Research Ethical Committee; CEP7: Centromere 7; CEP9: Centromere 9; CM: patients with histologic diagnosis of chagasic mega-esophagus; CONEP: Brazilian National Research Ethics Committee; DAPI: 4’, 6’-diamino-2-phenylindole;EGFR: epidermal growth factor receptor [erythroblastic leuke-mia viral (v-erb-b) oncogene homolog, avian]); ESCC: Esophageal squamous cell carcinoma; FFPE: formalin-fixed, paraffin-embedded;FGFR1:fibroblast growth fac-tor recepfac-tor 1;FHIT: fragile histidine triad gene; FISH: Fluorescence In Situ Hybridization; FITC: fluorescein isothiocyanate; HE: Hematoxylin and Eosin stain; LOH: loss of heterozygosity; MYC: v-myc myelocytomatosis viral oncogene homolog (avian);NCOA3:nuclear recep-tor coactivarecep-tor 3; NM: health individuals with histologi-cally normal esophagus; PIK3CA: phosphoinositide-3-kinase, catalytic, alpha polypeptide; Q-PCR: Quantitative - Polymerase Chain Reaction; SSC: Sodium chloride, sodium citrate solution; TP63:tumor protein p63; Vs: versus;YES1:v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1.

Acknowledgements

The authors are grateful to Dr. Kenji Miyazaki and Henrique Oliveira for collecting the biopsies; Dr. Patricia Maluf Cury for providing the tissue blocks and pathology evaluation; Dr. Sebastião Roberto Taboga and Luiz Roberto Faleiros for help with histological sections. We also thank the Brazilian Agency CAPES and the NCI grants U01CA85070, P30-CA46934 and P50 CA58187 for financial and technical support.

Author details

1

UNESP, São Paulo State University, Department of Biology, Campus São José do Rio Preto, SP, Brazil.2University of Colorado Denver, Department of Medicine/Medical Oncology, Aurora, Colorado, USA.3UNESP, São Paulo State University, Department of Computer Sciences and Statistics, Campus São José do Rio Preto, SP, Brazil.

Authorscontributions

MFB carried out the probe development, molecular cytogenetic studies and drafted the manuscript. AJM performed the statistical analysis. AES and MVG conceived the study, participated in its design and execution and contributed to the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 21 July 2009

Accepted: 17 February 2010 Published: 17 February 2010

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(10)

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Pre-publication history

The pre-publication history for this paper can be accessed here:http://www. biomedcentral.com/1471-230X/10/20/prepub

doi:10.1186/1471-230X-10-20

Cite this article as:Belliniet al.:Chromosomal imbalances are

uncommon in chagasic megaesophagus.BMC Gastroenterology2010

10:20.

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Figure

Table 1 Mean copy number per cell for the 12 DNA targets tested in esophageal mucosa of chagasic megaesophagusand normal mucosa groups.
Table 3 Comparison between matched gene andcentromere targets, in chagasic megaesophagus andnormal mucosa groups.
Figure 1 Exploratory analysis using three-dimensional plots of the percentages of genes status for individual cases is illustrated
Figure 2 FISH images for the unbalanced genes and centromeric targets in the megaesophagus specimens

References

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